DIFFERENTIAL LOCALIZATION PATTERNS OF MYRISTOYLATED AND NONMYRISTOYLATED C-SRC PROTEINS IN INTERPHASE AND MITOTIC C-SRC OVEREXPRESSER CELLS

Myristoylation of pp60src is required for its membrane attachment and transforming activity. The mouse monoclonal antibody, mAb327, which re cognizes both normal, myristoylated pp60c-src and a nonmyristoylated m utant, pp60c-src/myr-, has been used to compare the effects of prevent ing myristoylation on the localization of c-Src in NIH 3T3-derived ove rexpresser cells using immunofluorescence microscopy. During interphas e, pp60c-src partitions between the plasma membrane and the centrosome , while pp60c-src/myr is predominantly cytoplasmic but also partly nuc lear. The cytoplasmic, but not the nuclear, staining can be readily wa shed out by brief pretritonization of the cells before fixation, indic ating that the cytoplasmic pool of pp60c-src/myr-, in contrast with th e nuclear one, does not associate tightly with structures that are ins oluble in the presence of nonionic detergents. We have previously show n that during G2 phase, pp60c-src leaves the plasma membrane and is re distributed diffusely throughout the cytoplasm and to two clusters of patches surrounding the two separating centriole pairs. In contrast, w e now find that pp60c-src/myr- translocates to the nucleus in late G2 or early prophase prior to there being any clear evidence of nuclear m embrane breakdown or nuclear lamina disassembly. Similar nuclear trans location of pp60c-src/myr- but not of pp60c-src, is also observed when cells are arrested in Go or at the G1/S transition. Furthermore, duri ng mitosis, pp60c-src is found primarily in diffuse and patchy structu res dispersed throughout the cytoplasm while pp60c-src/myr- more speci fically associates with the main components of the spindle apparatus ( poles and fibers) and inside the interchromosomal space. These results suggest that a possible role for myristoylation might be to prevent u nregulated nuclear transport of proteins whose nonmyristoylated counte rparts are readily moved into the nucleus. They also raise the possibi lity that a subfraction of wild-type pp60c-src may behave, at specific times, like its nonmyristoylated counterpart, and may translocate to the nucleus and exert specific functions in that location.